Evaluation of Chondroprotective Nutriceuticals in an in Vitro Osteoarthritis Model

Introduction: Controversy remains over mechanisms by which nutriceuticals may lead to modulation of disease symptoms and cartilage degradation in osteoarthritis (OA), and which product is preferred for treatment of OA. Therefore, the purpose of this study was to evaluate the effects of glucosamine and chondroitin sulfate on chondrocyte viability, extracellular matrix production, markers of inflammation and degradation, and gene expression of anabolic and catabolic factors in an in vitro model of OA. Our study hypothesis was that glucosamine and chondroitin sulfate would significantly retard adverse effects on articular cartilage health associated with IL-1ß in a co-culture (cartilage and synovium explants) model of OA. Methods: Full-thickness articular cartilage and synovium explants were obtained from canine cadavers (n=5, mean weight 24.4±2.4 Kg) after euthanasia performed for reasons unrelated to this study. Four-mm cartilage and 6-mm synovial discs (n=42/each dog, respectively) were prepared, and synovium explants were placed in 24-well plates with one explant/well. Filter inserts (0.4μm pore) were placed in the culture plate and paired cartilage explants were placed in the filter inserts with one explant/insert. Explants were cultured in 1ml of RPMI1640 containing 10% FBS and antibiotics with various concentrations of glucosamine (G) (422μg/ml, 211μg/ml, or 105μg/ml), chondroitin sulfate (C) (42μg/ml, 21μg/ml, or 10.5μg/ml), or their mixture (G+C) (463μg/ml, 231μg/ml, or 115μg/ml) (IAMS Co.) in the presence of recombinant human IL-1ß (100ng/ml) (R&D Systems Inc.). Explants cultured without either nutriceutical or IL-1ß served as controls, and those without either nutriceutical, but with IL-1ß, were recognized as the IL group. The liquid media were refreshed and saved every 3 days and explants were collected on days 1, 3, and 12. Liquid media were used for evaluation of concentration of nitric oxide by Griess assay, prostaglandin E2 (P GE2) by enzymeimmunoassay, and MMP3 by ELISA. Cartilage explants (total 84 samples, n=2/each group/each sample collection time) were used for quantitative gene expression analysis for extracellular matrix components (C2: collagen II, C1: collagen I, Ag: aggrecan, De: decorin), proteinases (M1: MMP1, M3: MMP3, M13: MMP13, A1: aggrecanase1, A2: aggrecanase2), proteinase inhibitors (T1: TIMP1, T2: TIMP2, T3: TIMP3), and inflammatory mediators (NO: iNOS and CX: COX-2) by real time RTPCR. The other cartilage explants (total 126 samples, n=3/each group/each sample collection time) were used for evaluation of cell viability (Live/Dead assay, Molecular Probes Inc.), water content, and matrix content (GAG: glycosaminoglycan and HP: hydroxyproline). Results: Water content – There were no significant differences in water content among groups at any sample collection time, suggesting none of the treatments resulted in significant edema (malacia) or dehydration (dessication) of the cartilage explants. Cell viability – The majority of chondrocytes in cartilage explants emitted green fluorescence (live cell signal) at days 1 and 3 regardless of treatment group, while the number of red fluorescent chondrocytes (dead cells) were subjectively highest in the IL group at day 12 (Fig.1). Objective image analysis using image analysis software (Image-Pro Plus, MediaCybernetics) revealed that the IL group had significantly (P<0.05) lower cell viability than all others on day 12, except the lowest concentratio ns of C and G+C groups, indicating nutriceuticals tested prevented cell death associated with IL1ß in this model. Matrix content – The results of DMMB assay showed that GAG was well preserved in those cartilage explants cultured with G422μg, C21μg, and C10.5μg groups. However, there were no significant differences compared to controls or the IL group. Total collagen content of cartilage explants as determined by measuring HP was not significantly different in any treatment group compared to controls or the IL group. Gene expression – Gene expression of matrix macromolecules and proteinase inhibitors in the IL group were lower than controls while those of proteinases and inflammatory mediators in the IL group were higher than controls. Significant (p<0.001) differences in gene expression compared to the IL group were expressed as follows: ? : less than 10 fold increase, ??: less than 100 fold increase, ???: less than 1000 fold increase, ????: more than 1000 fold increase, ? : less than 10 fold decrease, ??: less than 100 fold decrease, ???: less than 1000 fold decrease, ????: more than 1000 fold decrease, and ? : no differences (Table 1). Although the results varied among treatment groups and over time, in general, G+C retarded inhibition of matrix expressio n and TIMP expression while G or C retarded elevation of proteinases and inflammatory mediators associated with the IL-1ß. Media analysis – Nitric oxide concentrations in the IL groups were consistently higher than controls throughout the study period, however, no statistical differences were present among groups. PGE2 concentrations were significantly (p<0.05) different among groups at each sample collect ion time, and G and C groups retarded PGE2 elevation associated with IL-1ß (Fig. 2). Despite significant alterations in MMP3 gene expression compared to IL, MMP3 concentrations in conditioned media were not significantly different among groups.